Tripartite efflux pumps enable Gram-negative bacteria to extrude diverse toxins, contributing to bacterial multidrug resistance and the emerging threat of untreatable bacterial infections. These efflux pumps require the assembly of an inner-membrane pump, a periplasmic adaptor protein, and an outer- membrane channel into a protein complex to extrude chemicals. In a collaboration, the applicant recently discovered that CusCBA, an RND-family metal efflux pump, undergoes dynamic assembly in response to cellular demands for metal efflux. Understanding such mechanisms of these efflux pumps and exploring novel methods to compromise their functions are crucial for developing new and effective antibacterial treatments. On the other hand, while the effects of chemical stressers, such as antibiotics, on bacterial physiology are well described, nothing is known about whether or how mechanical stresses may affect the assembly and function of tripartite efflux pumps such as CusCBA, even though mechanical forces are experienced in bacterial growth environments. The long-term goal of this research is to understand how bacterial efflux can be manipulated for preventive and therapeutic purposes. The objective here is to understand how mechanical stress can alter the assembly of CusCBA in live E. coli cells and thus cells? resistance to metal stress. The central hypothesis here, supported by preliminary studies, is that mechanical stress, by inducing cell deformations, can compromise the assembly of CusCBA in cells and thus their efflux function, making cells less resistant to metal stress. This hypothesis will be tested using combined approaches of single-molecule tracking, nanofluidics-based mechanical manipulations, chemical/genetic manipulations, and bulk biophysical/biochemical/cellular assays. The applicant will be advised by a mentoring team that includes a chemist with expertise in single-molecule imaging of bacterial metal efflux, a mechanical/biomedical engineer with expertise in mechanobiology, and a microbiologist. The rationale for this research is that, once it is accomplished, it will help devise mechanical strategies to impair the assembly of CusCBA and related tripartite efflux pumps and thus bacterial efflux to increase the efficacy of antibiotic treatments. The proposed research has two specific aims: 1) Define how mechanical stress alters CusCBA assembly and cells? resistance to toxic metals. 2) Identify the role of cell stiffness in coupling mechanical stress to CusCBA assembly in cells. The research is significant because it will advance the mechanobiology of bacterial efflux, the development of mechanical strategies to intervene in bacterial efflux for antibacterial therapy, and new technologies for mechanically manipulating single bacterial cells. It is innovative because it introduces the novel concept of mechano-efflux coupling and it uses the novel techniques of single-molecule tracking via time-lapse stroboscopic imaging and nanofluidic manipulation of individual bacterial cells.